The opportunistic pathogen Pseudomonas aeruginosa ranks among the leading causes of nosocomial infection. The type III secretion system (T3SS) aids acute Pseudomonas aeruginosa infection by injecting potent cytotoxins into host cells to suppress the host's innate immune response. Expression of all T3SS-related genes is strictly dependent on the transcription factor ExsA. Consequently, ExsA and the biological processes that regulate ExsA function are of great biomedical interest. The present study focused on the ExsA–ExsC–ExsD–ExsE signaling cascade, which ties host cell contact to the upregulation of T3SS gene expression. Prior to T3SS induction, the antiactivator protein ExsD binds to ExsA and blocks ExsA-dependent transcription by interfering with ExsA dimerization and promoter interactions. Upon host cell contact, ExsD is sequestered by the T3SS chaperone ExsC, resulting in the release of ExsA and upregulation of the T3SS. Previous studies have shown that the ExsD–ExsA interactions are not freely reversible. Because independently folded ExsD and ExsA were not found to interact, it has been hypothesized that folding intermediates of the two proteins form the complex. Here, we demonstrate, for the first time, that ExsD alone is sufficient to inhibit ExsA-dependent transcription in vitro and that no other cellular factors are required. More significantly, we show that independently folded ExsD and ExsA are capable of interacting, but only at 37 °C and not at 30 °C. Guided by the crystal structure of ExsD, we designed a monomeric variant of the protein, and demonstrated that ExsD trimerization prevents ExsD from inhibiting ExsA-dependent transcription at 30 °C. We propose that this unique mechanism plays an important role in T3SS regulation.